DE69534670T2 - IMMUNOSTIMULATING COMPOSITIONS CONTAINING A MINERAL SALT AND A FURTHER ADJUVANT - Google Patents

Abstract

Adjuvant compositions for modulating an immune response to an antigen administered to a host comprise a mineral salt adjuvant and at least one other adjuvant. The compositions provide an adjuvanting effect on an antigen which is greater than the adjuvanting effect attainable by one of the adjuvants alone. An antigen is covalently bonded to a glycolipid analog to provide a discrete molecule which exhibits an enhanced adjuvanting effect on the antigen which is greater than the adjuvanting effect attainable in the absence of such covalent bonding.

Description

AREA OF INVENTION

The The present invention relates to the field of immunology, and to them especially relates to adjuvants, d. h., materials that the Immune response to an antigen modulate.

BACKGROUND THE INVENTION

vaccines have been used for many years to humans and animals against a big one Variety of infectious Protect diseases. Such conventional Vaccines consist of attenuated ones Pathogenic (for example poliovirus), killed pathogens (for example Bordetella pertussis) or immunogenic components of the pathogen (for example diphtheria toxoid). Some antigens are in strong Measures immunogenic and alone are capable of protective To elicit immune responses. However, other antigens are not able to provide a protective Induce immune response or induce only a weak immune response.

at the development of some vaccines and immunogenic compositions There is a trend for smaller and well-defined immunogens and protective materials to use. recent Advances in molecular genetics, protein biochemistry, peptide chemistry and immunobiology have economic and efficient technologies provided to big Identify quantities of pure antigens from different pathogens and manufacture. Nevertheless, could some of these materials may not be sufficiently immunogenic, and either because of their small size (especially synthetic Peptides) or due to the lack of intrinsic immunostimulatory Properties of it.

The immunogenicity can be significantly improved if the antigens be administered together with adjuvants. Increase adjuvants the immunogenicity of an antigen, however, are not necessarily immunogenic themselves. Adjuvants can act by placing the antigen locally near the site of administration to produce a depot effect that causes a slow, long-lasting release of antigen to cells of the immune system facilitated. Adjuvants can also attract cells of the immune system to an antigen depot and stimulate such cells to elicit immune responses.

immunostimulatory Agents or adjuvants have been used for many years to treat the Immune responses of a host, for example, to improve against vaccines. Intrinsic adjuvants, such as lipopolysaccharides, are normal the components of the killed or used as vaccines attenuated Bacteria. Extrinsic adjuvants are immunomodulators which typically non-covalently bound to antigens and formulated to increase the immune responses of the host. Thus, adjuvants became Identifies the immune response to parenterally delivered antigens increase. However, some of these adjuvants are toxic and can cause unwanted side effects cause it unsuitable for use in humans and in many animals. In fact, only aluminum hydroxide and aluminum phosphate (collectively referred to as alum) routinely as adjuvants in human and veterinary Vaccines used. The effectiveness of Alumen in increasing of antibody responses on diphtheria and tetanus toxoids is generally confirmed, and Only recently was an HBsAg vaccine with alum as adjuvant added. Although the usability of Alumen for some Applications is well established, it has limitations. For example Alumen ineffective for influenza vaccination and calls a cell-mediated Immune response in an inconsistent manner. By antigens with alum antibodies produced as adjuvants are mainly of IgG1 isotype in the mouse, which is protected by some vaccine agents could not be optimal.

One greater Range of extrinsic adjuvants can potent immune responses to cause antigens. These include saponins that bind to membrane protein antigens Complexed (immune-stimulating complexes), Pluronicpolymere with mineral oil, killed Mycobacteria in mineral oil, complete friend's Adjuvant, bacterial products such as muramyl dipeptide (MDP) and lipopolysaccharide (LPS), as well as lipid A and liposomes.

To efficiently induce humoral immune responses (HIR) and cell-mediated immunity (CMI), immunogens are involved in adjuvants. Many adjuvants are toxic, inducing granuloma, acute and chronic inflammation (complete Freund's adjuvant, FCA), cytolysis (saponins and pluronic polymers) and pyrogenicity, arthritis and anterior uveitis (LPS and MDP). Although FCA an out is widely used in research, it is not licensed for use in human or veterinary vaccines because of its toxicity.

• (1) Mangel an Toxizität;
• (2) Vermögen, eine lang anhaltende Immunantwort zu stimulieren;
• (3) Einfachheit der Herstellung und Stabilität bei Langzeitlagerung;
• (4) Vermögen, sowohl CMI als auch HIR auf Antigene, welche über verschiedene Routen verabreicht werden, hervorzurufen, sofern erforderlich;
• (5) Synergie mit anderen Adjuvantien;
• (6) Vermögen des selektiven Wechselwirkens mit Populationen von Antigen präsentierenden Zellen (APC);
• (7) Vermögen, spezifisch geeignete T_H1- oder T_H2-Zell-spezifische Immunantworten hervorzurufen; und
• (8) Vermögen, selektiv geeignete Antikörper-Isotyp-Spiegel (zum Beispiel IgA) gegen Antigene zu steigern.

Desirable characteristics for ideal adjuvants include:

• (1) lack of toxicity;
• (2) ability to stimulate a long-lasting immune response;
• (3) ease of manufacture and stability in long-term storage;
• (4) ability to elicit both CMI and HIR on antigens administered via different routes, if necessary;
• (5) synergy with other adjuvants;
• (6) ability of selective interaction with populations of antigen presenting cells (APC);
• (7) ability to elicit specifically suitable T _H 1 or T _H 2 cell-specific immune responses; and
• (8) ability to selectively raise suitable antibody isotype levels (for example, IgA) to antigens.

The to Lockhoff et al. U.S. Patent No. 4,855 issued August 8, 1989 283 teaches glycolipid analogs, including N-glycosylamides, N-glycosyl ureas and N-glycosyl carbamates, each of which in the sugar residue by an amino acid substituted as immunomodulators or adjuvants. Thus, Lockhoff reported et al. (U.S. Patent No. 4,855,283) that N-glycolipid analogs have structural similarities across from the course occurring glycolipids, such as glycosphingolipids and glycoglycerol lipids, are able to show strong immune responses in both cases Herpes simplex virus vaccine as well as pseudorabies virus vaccine cause. Some glycolipids were made from long-chain alkylamines and fatty acids, which are directly linked to the sugars by the anomeric carbon atom, synthesizes the functions of naturally occurring lipid residues imitate.

The U.S. Patent No. 4,258,029 to Moloney assigned to the assignee thereof, teaches that octadecyl tyrosine hydrochloride (OTH) as a Adjuvant when used with tetanus toxoid and with formalin-inactivated Type I, II and III poliomyelitis virus vaccine is complexed. Also, Nixon-George et al. (1990) J. Immunology 144: 4798-4802, that octadecyl ester of aromatic amino acids containing a recombinant Hepatitis B surface antigen Complexed are the immune responses of the host to hepatitis B virus increase.

The Lipidation of synthetic peptides has also been used about their immunogenicity to increase. Thus Wiesmuller ((1989), Vaccine 7: 29-33) have a peptide with a sequence described to be a protein of foot-and-mouth disease virus homologous coupled to a tripalmityl S-glyceryl-cysteinylserylserine adjuvant, that is a synthetic analog of the N-terminal part of the lipoprotein of gram-negative bacteria. Furthermore, Deres et al. (1989, Nature 342: 561) in vivo priming of virus-specific cytotoxic T-lymphocytes with synthetic Lipopeptide vaccine containing modified synthetic peptides derived from the influenza virus nucleoprotein by the linkage a lipopeptide, N-palmityl S- [2,3-bis (palmityloxy) - (2RS) -propyl- [R] -cysteine (TPC) included.

The Adjuvants and immunostimulating compounds described above are, can no adjuvant capacity for all Provide antigens to a variety of hosts among many Conditions supplied become.

It would be desirable To provide adjuvant compositions which do not conform to the Disadvantages and limitations from currently available Adjuvants suffer.

Summary the invention

• (a) ein anorganisches Adjuvanssalz; und
• (b) wenigstens ein anderes Adjuvans, welches aus der Gruppe bestehend aus einem Glycosylamid, einem Octadecylester einer Aminosäure und einem Lipopeptid ausgewählt ist.

The present invention aims to provide improved adjuvant compositions. According to one aspect of the present invention, there is provided an adjuvant composition for modulating an immune response to an antigen administered to a host, the composition comprising:

• (a) an inorganic adjuvant salt; and
• (b) at least one other adjuvant selected from the group consisting of a glycosylamide, an octadecyl ester of an amino acid and a lipopeptide.

The multiple adjuvant compositions provided herein exhibit a surprisingly unexpected adjuvant effect on an antigen greater than the adjuvant effect achievable by one of the adjuvants alone. The enhanced effect can be additive to the Adjuvanseffekt the individual Ad may be adjuvants, and in particular embodiments a synergistic effect is achieved.

The inorganic adjuvant salt or mineral salt adjuvant preferably comprises Aluminum hydroxide or aluminum phosphate, although other known ones inorganic adjuvant salts, such as calcium phosphate, zinc hydroxide or Calcium hydroxide, can be used.

One Glycosylamide is a glycolipid analogue. The amino acid can an aromatic amino acid, for example tyrosine.

The Lipopeptide may be a synthetic analog of an N-terminal region a lipoprotein from a Gram-negative bacterium.

The Lipopeptide may be tripalmityl-S-glyceryl-cysteinylseryl-serine or N-palmityl-S- (2,3-palmityloxy) -2- (RS) -propyl- [R] -cysteine be.

worin R¹ Wasserstoff oder einen gesättigten oder einfach oder mehrfach ungesättigten Alkylrest mit bis zu 50 Kohlenstoffatomen darstellt;
X -CH₂-, -O- oder -NH- darstellt,
R² Wasserstoff oder einen gesättigten oder einfach oder mehrfach ungesättigten Alkylrest mit bis zu 50 Kohlenstoffatomen darstellt;
R³, R⁴ und R⁵ unabhängig voneinander Wasserstoff oder SO₄ ^2– oder PO₄ ^2– oder einen anderen negativ geladenen Rest oder -CO-R⁶ darstellt, wobei R⁶ ein Alkylrest mit bis zu 10 Kohlenstoffatomen ist;
R⁷ L-Alanyl, L-alpha-Aminobutyryl, L-Arginyl, L-Asparaginyl, L-Aspartyl, L-Cysteinyl, L-Glutamyl, L-Glycyl, L-Histidyl, L-Hydroxypropyl, L-Isoleucyl, L-Leucyl, L-Lysyl, L-Methionyl, L-Ornithinyl, L-Phenylalanyl, L-Prolyl, L-Seryl, L-Threonyl, L-Tyrosyl, L-Tryptophanyl und L-Valyl oder deren D-Isomere ist;
und pharmazeutisch verträgliche Salze davon.In a particular embodiment, the glycosylamide may have the following formula:

wherein R ^{1 represents} hydrogen or a saturated or mono- or polyunsaturated alkyl radical having up to 50 carbon atoms;
X represents -CH ₂ -, -O- or -NH-,
R ^{2 is} hydrogen or a saturated or mono- or polyunsaturated alkyl radical having up to 50 carbon atoms;
R ³ , R ⁴ and R ⁵ independently represent hydrogen or SO ₄ ^2- or PO ₄ ^2- or another negatively charged radical or -CO-R ⁶ wherein R ^{6 is} an alkyl radical of up to 10 carbon atoms;
R ^{7 is} L-alanyl, L-alpha-aminobutyryl, L-arginyl, L-asparaginyl, L-aspartyl, L-cysteinyl, L-glutamyl, L-glycyl, L-histidyl, L-hydroxypropyl, L-isoleucyl, L- Leucyl, L-lysyl, L-methionyl, L-ornithinyl, L-phenylalanyl, L-prolyl, L-seryl, L-threonyl, L-tyrosyl, L-tryptophanyl and L-valyl or their D-isomers;
and pharmaceutically acceptable salts thereof.

In an embodiment For example, the glycosylamide may be N- (2-deoxy-2-L-leucylamino-β-D-glucopyranosyl) -N-octadecyldodecanamide acetate be. Preferably, in this embodiment, the mineral salt Aluminum phosphate.

Preferably the composition further comprises at least one antigen for elicitation an immune response in a host.

Appropriate Antigens included in the immunogenic compositions can be and whereby an immune response is modulated include microbial Pathogens, bacteria, viruses, proteins, glycoproteins, lipoproteins, Peptides, glycopeptides, lipoproteins, toxoids, carbohydrates and tumor-specific Antigens. Mixtures of two or more antigens can be used for Application come.

Such Peptides, glycopeptides or lipopeptides may have an amino acid sequence lock in, those of an antigenic determinant of HIV, rubella virus, respiratory Syncytial virus, Bordetella pertussis, Haemophilus influenzae or Streptococcus pneumoniae corresponds, including those specific synthetic Peptides shown below in Table 1 (SEQ ID NO: 1 to 15) (the tables appear at the end of the descriptive text) or a functional analogue thereof. The toxoid may be a pertussis toxoid whereas the protein is influenza haemaglutinin or a subunit a human parainfluenza virus, such as the HN or F proteins of PIV-3, can be.

According to a second aspect of the invention there is provided an adjuvant composition, which is by an inorganic adjuvant salt and at least one other adjuvant selected from the group consisting of a glycosylamide, an octadecyl ester of an amino acid and a lipopeptide, for use in modulating an immune response to an antigen administered to a host.

According to one The third aspect of the invention features the use of an adjuvant composition by an inorganic adjuvant salt and at least one other Adjuvant selected from the group consisting of a glycosylamide, an octadecyl ester of an amino acid and a lipopeptide, in the manufacture of a drug with an antigen to one Immune response to an antigen administered to a host, to modulate.

• (a) Mittel, die ein anorganisches Adjuvanssalz enthalten;
• (b) Mittel, die wenigstens ein anderes Adjuvans enthalten, das aus der Gruppe, bestehend aus einem Glykosylamid, einem Octadecylester einer Aminosäure und einem Lipopeptid, ausgewählt ist;
• (c) Mittel, die wenigstens ein Antigen enthalten; und
• (d) Mittel zum Kombinieren des anorganischen Adjuvanssalzes, wenigstens eines anderen Adjuvans und wenigstens eines Antigens, zur Herstellung der immunogenen Zusammensetzung.

According to a fourth aspect of the invention there is provided a kit for the preparation of an immunogenic composition comprising:

• (a) agents containing an inorganic adjuvant salt;
• (b) means containing at least one other adjuvant selected from the group consisting of a glycosylamide, an octadecyl ester of an amino acid and a lipopeptide;
• (c) agents containing at least one antigen; and
• (d) means for combining the inorganic adjuvant salt, at least one other adjuvant and at least one antigen, to produce the immunogenic composition.

The achieved immune response can be a humoral or a cell-mediated Be immune response.

By covalent binding of an antigen to a glycosylamide becomes a single molecule which produces a surprising shows unexpected increased adjuvant effect on the antigen, which is larger than the Adjuvanseffekt is, which in the absence of such a covalent Binding is achievable, as in a mixture of the two components. Another enhanced adjuvant effect may be for such a covalently bonded one Antigen can be achieved by adding an inorganic adjuvant salt in such compounds is introduced.

The antigen may be linked to the glycosylamide at a carboxy or amino terminus or other suitable site compatible with covalent linking of the antigen, for example, by a crosslinker having a reactive function such as maleimidyl, succinimidyl, 2-pyridyldithio, NH ₂ , SH, and -CO-R8, wherein R8 is -OH, N _3, -O-alkyl (C ₁ -C _2), -OC ₆ F _5, H, Br or Cl, can be covalently bound.

• (a) Leichtigkeit der Formulierung;
• (b) Wirksamkeit des Adjuvansverhaltens; und
• (c) Kompatibilität von Antigenen mit der Adjuvanszusammensetzung.

Advantages of the present invention include:

• (a) ease of formulation;
• (b) effectiveness of adjuvant behavior; and
• (c) compatibility of antigens with the adjuvant composition.

BRIEF DESCRIPTION OF THE DRAWINGS

The 1 shows antibody responses to HIV peptides in guinea pigs formulated with adjuvants according to one embodiment of the invention;

the 2 Figure 4 shows antibody responses in guinea pigs to a cocktail of HIV peptides formulated with adjuvants according to one embodiment of the present invention;

the 3 shows haemagglutinin inhibition antibody responses in mice to a human parainfluenza virus subunit vaccine formulated with adjuvants according to the present invention;

the 4 shows virus neutralization antibody responses to a human parainfluenza virus vaccine subunit vaccine formulated with adjuvants according to the present invention.

The 5 shows guinea pig immune responses to an influenza split vaccine formulated with adjuvants according to one embodiment of the present invention against three influenza virus strains;

the 6 shows guinea pig immune responses (determined by anti-PT ELISA) to Pertusssi-To xoid formulated with adjuvants according to an embodiment of the present invention;

the 7 Figure 8 shows guinea pig immune responses (as determined by the CHO cell neutralization assay) (Grunstrom et al 1985 J. Infec. Dis 151: 646-649) for pertussis toxoid formulated with adjuvants according to one embodiment of the present invention;

the 8th shows a guinea pig immune response to an HIV peptide CLTB36 formulated with adjuvants or conjugated to BAY R1005 according to one embodiment of the present invention; and

the 9 shows guinea pig antibody responses to HIV peptide (CLTB 36) formulated with alum or conjugated to BAY R1005 according to one embodiment of the present invention.

GENERAL DESCRIPTION THE INVENTION

It will be apparent to those skilled in the field that different embodiments The present invention has many applications in the fields of Medicine and in particular in vaccination, diagnosis, generation of immunological agents and the treatment of infections with pathogens, including Bacteria and viruses possess. Another non-limiting discussion such uses will be further explained below.

As As noted above, the present invention relates in one aspect Adjuvant compositions useful for modulating the immune response are useful on an antigen.

synthetic Including antigens Vaccines, can produced by chemically synthesizing peptides, which share antigenic determinants with proteins, for example of HIV-1, rubella virus, RSV, Haemophilus influenzae type b, Bordetella pertussis and Streptococcus pneumoniae or other antigens. These peptides, lipid derivatives of such peptides, as well as viral antigens or bacterial antigens can either used individually or in combination as a cocktail and formulated with synthetic adjuvants and mineral salts to provide an immunogenic composition. These Compositions can used to mammals, for example by intramuscular or parenteral routes, or by delivery to mucosal surfaces below Use of microparticles, capsules, liposomes and targeted molecules like toxins and antibodies, to immunize.

It will now be described in detail to the presently preferred embodiments of the invention, which together with the following Examples help to explain the invention.

Anti gene selection

Several Antigens were chosen to exemplify the present invention. Progress in biotechnology it now to identify bacterial and viral antigens and in a large scale too clean. However, subunit are present or synthetic Vaccine candidates sometimes due to low immunogenicity their size (in particular synthetic peptides) or lack of intrinsic immunostimulatory Properties. Thus, they are common external additives needed to increase their immunogenicity. Several Antigens are chosen which are capable of producing strong IgG antibody responses in adjuvants, like CFA, to evoke. The chosen ones Close antigens synthetic peptides (Table 1), which are antigenic determinants with the proteins of HIV-1, rubella virus (RV), respiratory Syncytial virus (RSV), Haemophilus influenzae type b (Hib), Bordetella pertussis and Streptococcus pneumoniae and the HN and F proteins from the parainfluenza virus 3 (PIV3), pertussis toxoid and chemically disrupted influenza virus.

Synthetic adjuvants

Synthetic adjuvants such as glycolipid analogs (Lockhoff et al., U.S. Patent No. 4,855,283), lipopeptide (Wiesmuller et al. (1989), Vaccine 7: 29-33) and octadecyl esters of aromatic amino acids (Moloney et al., US Pat Patent No. 4,258,029) have been shown to act as adjuvants, increasing the immunogenicity of viral and bacterial antigens. Therefore, three adjuvants, namely N-palmityl S- [2,3-bis (palmityloxy) - (2RS) -propyl- [R] -cysteine (TPC, Wiesmuller et al., Vaccine (1989) 8: 29-33 ); N- (2-Deoxy-2-L-leucylamino-β-D-glucopyranosyl) -N-octadecyldodecanamide acetate (BAY R1005, O. Lockhoff, Angew Chem .. Int Ed. Engl. (1991) 30: 1611-1620) ; and Octadecyl tyrosine (OTH) (Nixon-George et al. (1990), J. Immunology 144: 4798-4802) as starting molecules for the design of more potent synthetic adjuvant chosen. These three classes of synthetic adjuvants were synthesized and characterized. The synthesis of the three classes of adjuvants requires less than 10 reaction steps. The protocols for adjuvant synthesis are well established and described in the literature. The scale-up production for BAY R1005 and octadecyl tyrosine would be within the skill of the art.

All Three classes of synthetic adjuvants are in water or aqueous Buffer, such as buffered phosphate salt solution (PBS) insoluble. she form a milky solution, if you are using water or aqueous buffer be mixed. They are non-toxic, as by the absence of adverse Reactions in mice which 1 to 2 mg were injected, and they are at Non-pyrogenic rabbit pyrogen test. All three classes of synthetic Adjuvants are in powdered form Shape very stable at -20 ° C and can in watery Buffer for a long-term storage at 4 ° C be suspended.

Around to analyze the effectiveness of synthetic adjuvants 100 μg synthetic Peptides (Table 1), which known functional T helper and B cell epitopes contain, used as antigens. Fifteen peptides, including Epitopes of HIV-1, RSV, rubella virus, H. influenzae, B. pertussis and S. pneumoniae were single intramuscularly in guinea pigs either in the presence of FCA or alum (aluminum phosphate) or TPC or BAY R1005 or OTH or PBS injected. The effect of adjuvants on Antibody responses was evaluated using peptide-specific ELISAs. As shown in Table 2 below, all in FCA emulsified Peptides strong antibody responses peptides in PBS are either only very low Anti-peptide titers, or failed, any detectable Antibody response cause. After three immunizations, the fifteen were on Alums absorbed peptides capable of peptide-specific IgG antibody responses to induce. Only two peptides (RV-EP27 and PSP-AA) in the presence BAY R1005 failed to provide significant antibody responses to produce three immunizations. One of the peptides (RV-EP27), which failed to give significant antibody responses in BAY R1005 was highly immunogenic and induced peptide-specific antibody responses, when TPC was used as adjuvant. In most cases increased both synthetic adjuvants, TPC and OTH, the immunogenicity of the peptide, however, the reactive titers were much lower than those which from almumen or CFA. These results are with the published Data published by other working groups consistently, and they show the adjuvant potential of the synthetic adjuvants and the capacity of these peptides to be enhanced by adjuvants.

In one aspect, the present invention provides an adjuvant composition for modulating an immune response to an antigen administered to a host, the composition comprising an inorganic adjuvant salt and at least one other adjuvant. To exemplify this aspect of the invention, several HIV-1 peptides have been used in guinea pig immunogenicity studies. Peptides were first absorbed onto alum and then emulsified with the synthetic adjuvant before being injected into different groups of guinea pigs. Guinea pigs were also immunized with either alum-absorbed peptides or synthetic adjuvant-emulsified peptides as controls. The antibody response results obtained with the synthetic adjuvant BAY R1005 are in the 1 shown. No adverse reactions were found in any of the guinea pigs. It has surprisingly been found, according to the present invention, that alum (3 mg / ml) combined with a synthetic adjuvant (BAY R1005 (1 mg / ml)) resulted in increased anti-peptide antibody responses after two immunizations, in the Comparison to those titres elicited by the same peptide using either synthetic adjuvant or mineral salts alone as adjuvant. These studies thus show that the immune response to an antigen which has already been increased by absorption on alum under standard conditions can unexpectedly be further enhanced by another adjuvant according to the present invention.

The unexpected immune-enhancing capital Adjuvant compositions of the present invention have been further developed for the synthetic peptides RSV-F and RV-EP27 (Table 1). If those Peptides supplemented with alum alone as an adjuvant were antibody titers from 32,000 (RSV-F) and 12800 (RV-EP27) (Table 3). If the same peptides were adjuvanted with BAY R1005, they were only a little immunogenic. However, when these peptides with an adjuvant composition were adjuvanted, which included alum and BAY R1005 antibody titers of 128000 (RSV-F) and 64000 (RV-EP27). These results thus show the capacity this adjuvant composition, the immune response to an antigen to increase, which is beyond that obtainable by a single adjuvant thereof. In fact, an immune response was obtained which was more than that Sum of the immune response to each of the adjuvants was single, d. h., a synergistic effect was obtained.

To further evaluate the adjuvant effect of the present invention, parainfluenza virus 3 (PIV3) HN and F glycoproteins were first absorbed onto alum and then emulsified with BAY R1005 before being injected with interperitoneal (ip) CD1 mice. As controls, the same antigens were either absorbed on alum or emulsified with BAY R1005 or mixed with PBS and then used to immunize ip groups of CD1 mice. No adverse reactions were found in any of the mice. It was surprisingly found that the primary antibody responses to HN and F were best with the Alumene / BAY R1005 adjuvant composition. The functional antibody responses, as measured by hemagglutinin inhibition and virus neutralization assays, revealed that with antigen formulated with an adjuvant composition of the present invention, immunized mice produced higher antibody levels than obtained with a single adjuvant ( 3 and 4 ).

To determine if the steps of the formulation would affect the adjuvant effects of the compositions of the present invention, commercially available flu split vaccine was first emulsified with BAY R1005 (1 mg / ml) and then absorbed on alum (3 mg / ml). before being injected intraperitoneally (ip) to mice. As controls, the same preparation was either absorbed on alum or emulsified with BAY R1005 or mixed with PBS and then used to immunize different groups of mice. Again, no adverse reactions were detected in any of the groups of mice. The BAY R1005 / Alumen Combination Adjuvant Formulation gave the best anti-HA antibody responses ( 5 ) against various influenza strains.

The Results obtained with the split feline vaccine indicate that the sequence of steps leading to the formulation of the antigen with different adjuvants were used, not essential for the obtained Adjuvanseffekt is.

The adjuvant compositions of the present invention were also able to modulate the immune response to gluteraldehyde-inactivated pertussis toxoid. Thus guinea pigs were immunized with pertussis toxoid in alum alone, BAY R1005 or BAY R1005 anti-PT, and titers of toxin neutralizing antibodies were determined ( 6 and 7 ). The presented results indicate that the highest (and a synergistic) immune response was obtained when the pertussis toxoid was formulated with alum and BAY R1005 according to the present invention.

Immunogenicity of peptides, which are covalently bound to synthetic adjuvants

The task of providing a synthetic peptide as a self-sufficient immunogen capable of eliciting both humoral and cell-mediated immune responses is very demanding. To determine whether a peptide antigen covalently linked to an adjuvant can elicit both a humoral and a cell-mediated immune response, the CLTB-36 peptide was synthesized with synthetic adjuvant BAY R1005 covalently attached to the N-terminus. During the production of CLTB-36 covalently linked to BAY R1005, it may be desirable to use Fmoc peptide synthetic chemistry and to temporarily protect reactive functional groups, for example, alcohol through t-butyl and acids through ester groups , Suitable contactor deprotection conditions and protocols are described herein in the Examples. The peptide-adjuvant conjugate was purified by RP-HPLC and used to immunize guinea pigs. These immunogenicity studies revealed that CLTB-36 covalently linked to BAY R1005 was as immunogenic as CLTB-36 formulated with (BAY R1005 + alumene) ( 8th . 9 and Table 4). The anti-CLTB-36 antibody titer obtained was about 3-fold and 20-fold higher than those elicited in the presence of either BAY R1005 or alum ( 8th ). In addition, the peptide-adjuvant conjugate (BAY-CLTB-36) required fewer immunizations and less antigen to elicit the same level of anti-peptide antibody responses as in the US Pat 9 shown. These results indicate that an antigen with a built-in immunomodulator can induce strong immune responses.

Vaccine production and -Use

As indicated above, in one embodiment, the present invention provides adjuvant mixtures useful for formulating immunogenic compositions suitable for use, for example, as vaccines. The immunogenic composition elicits an immune response by the host to which it is administered, including the production of antibodies by the host. The immunogenic compositions include at least one antigen in one embodiment. This antigen may be an inactivated pathogen or an antigenic fraction of a patho be his. The pathogen may be, for example, a virus, a bacterium or a parasite. The pathogen may be inactivated by a chemical agent such as formaldehyde, glutaraldehyde, β-propiolactone, ethyleneimine and derivatives, or other compounds. The pathogen may also be inactivated by a physical means such as UV radiation, gamma radiation, "thermal shock" and X-radiation.

A antigenic fraction of a pathogen can be detected with the help of chemical or physical decomposition processes are produced, followed, if desired, from the separation of a fraction by means of chromatography, Centrifugation or similar Techniques. Generally, low molecular weight components then become which, while purified, have low immunogenicity can. Alternatively you can Antigens or haptens with the help of organic synthetic methods or in the case of, for example, polypeptides and Proteins using recombinant DNA methods.

vaccines which contain peptides are well known in the art, as described by U.S. Patents 4,601,903; 4,599,231; 4,599,230 and 4,596 792 exemplified; all references hereof the reference is included.

The Use of peptides in vivo may be their first chemical modification since the peptides themselves do not have sufficiently long serum and / or tissue half-life and / or sufficient immunogenicity.

For this Purpose can be the molecule optionally bound to a carrier molecule of the invention become. Many suitable links are known, for example, the use of the side chains of the Tyr residues. Suitable carriers shut down for example limpet hemocyanin (KLH), serum albumin, purified Protein derivative of tuberculin (PPD), ovalbumin, non-protein carriers and many others.

Furthermore It may be advantageous to modify the peptides to them a conformational restriction impose. This can be useful to be, for example, a natural one occurring conformation of the peptide in the context of the native protein mimic the effector immune responses that they elicit, to optimize.

modified Peptides are referred to herein as "peptide analogs". Of the Expression "peptide analog" includes any functional chemical equivalent a peptide, which by its increased stability and / or effectiveness and immunogenicity characterized in vivo or in vitro with respect to the practice of the invention is. The term "peptide analog" is also used herein used to refer to any amino acid derivative of the peptides, such as as described herein. Herein into consideration drawn peptide analogs are prepared by procedures which Modifications to side chains, the introduction of unnatural amino acids and / or their derivatives during peptide synthesis and the use of crosslinkers and others Methods which give the peptides or their analogues a conformational restriction imposing, including, but not limited to this are.

It will be appreciated that the peptides used herein as antigens can be modified in a variety of different ways without significantly affecting the functionally important immunogenic behavior thereof. Possible modifications to the peptide sequence include the following:
One or more individual amino acids may be substituted by amino acids having comparable or similar properties, such as:
V can be substituted by I;
T can be substituted by S;
K can be substituted by R, or
L can be substituted by I, V or M.

A or more of the amino acids of peptides of the invention replaced by a "retroinverso" amino acid be, d. h., By a bifunctional amine with a functional Group that is an amino acid corresponds, as in the published international application WO 91/13909.

A or more amino acids can be deleted.

structural Analogs that mimic the 3-dimensional structure of the peptide, can be used in place of the peptide.

Examples of side chain modifications contemplated by the present invention include modifications of amino groups, such as by reductive alkylation by reaction with an aldehyde, followed by reduction with NaBH ₄ ; amidation with methyl acetimidate; acetylation with acetic anhydride; the carbamylation of amino groups with 2,4,6-trinitrobenzene sulfonic acid (TNBS); the alkylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal 5'-phosphate, followed by reduction with NaBH ₄ .

The Guanidino group of arginine residues may be due to the formation of heterocyclic Condensation products with reagents such as 2, 3-butanedione, phenylglyoxal and glyoxal.

The Carboxyl group can be activated by carbodiimide activation via a o-acylisourea formation, followed by subsequent derivatization, For example, to a corresponding amide, be modified.

sulfhydryl can modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; of the Formation of mixed disulfides with other thiol compounds; the reaction with maleimide; Maleic anhydride or other substituted maleimide; the formation of mercury derivatives using of 4-chloro-mercuric-benzoate, 4-chloro-mercuric-phenylsulfonic acid, phenylmercuric chloride, 2-Chloro-mercury-4-nitrophenol and other mercury compounds; carbamylation with cyanate at alkaline pH.

tryptophan residues can for example, by the oxidation with N-bromosuccinimide or the alkylation of the Indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulfonyl halides be modified. Tryosine residues can be prepared by nitration with tetranitromethane changed to form a 3-nitrotyrosine derivative.

The Modification of the imidazole ring of a histidine residue can be achieved by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate become.

Examples for the Installation of unnatural amino acids and derivatives during close to the peptide synthesis the use of norleucine, 4-aminobutyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, Norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienylalanine and / or D-isomers of amino acids but are not limited thereto.

The immunogenic compositions are prepared as injections, such as liquid solutions or emulsions. The Antigens and immunogenic compositions may be treated with physiologically acceptable carriers be mixed, which are compatible with it. These can be water, Saline, Dextrose, glycerol, ethanol and combinations thereof. Of the Vaccine may also contain auxiliary substances, such as wetting agents. or emulsifiers or pH buffering agents to further their effectiveness to increase. vaccines can by subcutaneous or intramuscular Be administered injection.

alternative can according to the present Invention formed immunogenic compositions in a manner formulated and supplied to elicit an immune response to mucosal surfaces. Thus, the immunogenic composition can be administered to mucosal surfaces for example, through nasal or oral (intragastric) routes. Alternatively, other modes of administration, including suppositories, desirable.

For suppositories can Binder and carrier for example, polyalkylene glycols and triglycerides. oral Formulations can normally used inoculants, such as saccharin, cellulose and magnesium carbonate of pharmaceutical grade.

These Compositions can the form of solutions, Suspensions, tablets, pills, capsules, sustained release formulations or powders and 1 to 95% of the immunogenic compositions of the present invention.

The immunogenic compositions are administered in a manner compatible with the dosage formulation and in such amount as to be therapeutically effective, protective and immunogenic. The amount to be administered depends on the subject / subject to be immunized, including, for example, the ability of the patient / subject's immune system to synthesize antibodies and, if necessary, eliciting a cell-mediated immune response. The exact amounts of antigen and immunogenic composition to be administered will depend on the judgment of the practitioner. However, suitable dosage ranges are readily available to those in the art Experienced determinable and may be on the order of micrograms to milligrams. Suitable regimens for initial administration and booster doses are also variable, but may include an initial administration followed by subsequent administrations. The dosage of the vaccine may also depend on the route of administration and will vary according to the size of the host.

The Concentration of the antigen in an immunogenic composition according to the invention is generally 1 to 95%. A vaccine which is antigenic Containing material from only one pathogen is a monovalent vaccine. Vaccines containing antigenic material from several pathogens, are combined vaccines and also belong to the present Invention. Such combined vaccines include, for example Material from different pathogens or from different strains of the same pathogens, or combinations of different pathogens.

immunoassays

In an embodiment are the adjuvant mixtures of the present invention for the preparation of useful in immunogenic compositions which are used can, to antigen-specific antibodies which in the specific identification of the antigen in an immunoassay according to a diagnostic embodiment are usable. Such immunoassays include enzyme-linked immunosorbent assays (ELISA), RIAs and other non-enzyme-linked antibody binding assays or in the field known procedures. In ELISA assays, the antigen-specific antibody a selected one surface immobilized; for example the wells of a microtiter plate made of polystyrene. After washing to remove not fully adsorbed antibodies may be a nonspecific protein, such as a solution of bovine serum albumin (BSA) or casein, which for it is known to be antigenically neutral with respect to the test sample, at the chosen surface be bound. this makes possible blocking non-specific adsorption sites on the immobilizing surface and thereby reduces the non-specific binding of Antigens on the surface caused background. The immobilizing surface becomes then with a sample, such as clinical or biological materials, which in a for an immune complex (antigen / antibody) formation appropriate way to test, brought into contact. This can diluting the sample with diluents, such as BSA, bovine gamma globulin (BGG) and / or phosphate-buffered saline solution (PBS) / Tween. The sample is then during about 2 to 4 hours at temperatures, such as in the range of about 25 up to 37 ° C, allow to incubate. After incubation, take the sample contacted surface washed to remove non-immunocomplexed material. The Washing procedure may be washing with a solution such as PBS / Tween or a Borate buffer, include.

To the formation of specific immune complexes between the antigen in the test sample and the bound antigen-specific antibodies and a subsequent Washing may occur, and even the amount of immunocomplex formation, be determined by the immune complex with a second antibody specificity for the Antigen is exposed. To provide a detection means, may be the second antibody an associated activity, like an enzymatic activity, which, for example, a color development during incubation with a suitable chromogenic substrate. The quantification can then be achieved by reducing the degree of color generation below Use for example one working in the visible spectrum Spectrophotometer is measured. In an additional embodiment includes the present invention a diagnostic kit, which antigen-specific antibodies by immunizing a host with immunogenic Compositions according to the present invention Produced according to the invention.

EXAMPLES

The The above description generally describes the present invention. A more complete one understanding can be obtained by reference to the following specific examples become. These examples are for the purpose of illustration only and are not intended to limit the scope of the invention. amendments in the form and the substitution of equivalents are considered pulled, as the circumstances suggest or make it appear appropriate. Although specific expressions should be used herein, such terms in a be descriptive sense and not for the purpose of limitation.

example 1

This Example describes the preparation of synthetic peptides.

peptides (Table 1) were generated using an ABI 430A peptide synthesizer synthesized and optimized by t-Boc chemistry, as it is by the Manufacturer, and then from the resin by hydrofluoric acid (HF) cleaved. The peptides were purified by reverse phase high performance liquid chromatography (RP-HPLC) on a semi-preparative Vydac C4 column (1 × 30 cm) using a 10 to 50% acetonitrile gradient in 0.1% trifluoroacetic (TFA), which is about 40 minutes, purified at a flow rate of 2 ml / min. All synthetic peptides (Table 1) were> 95% pure, as by analytical HPLC assessed. Amino acid composition analysis these peptides, which were performed on a Waters Pico-Tag system, were in good agreement with their theoretical compositions.

Example 2

This Example describes the synthesis of adjuvants.

N-alkyl-N-glycosyl-carboxamide (N- (2-L-leucine-amino-2-deoxy-β-D-glucopyranosyl) -N-octadecyl-Dodecansäureamid, BAY R1005 and its analogs) were prepared according to the method which is described in Lockhoff et al., U.S. Patent No. 4,855,283 is. BAY R1005 and its analogues can be prepared by ion exchange chromatography be further cleaned. The octadecyl ester of an aromatic amino acid (OTH) became according to Moloney et al., U.S. Patent No. 4,258,029. Fmoc-S- (2,3-bis (palmitoyloxy) - [2R] propyl) - [R] -cysteine using the coupling procedure of Fmoc chemistry to one growing peptide chain and then at the N-terminus by a palmityl group acylated. TPC derivatives can either tripalmityl-Cys-Ser-Ser-Asn-Ala (Ad) or tripalmityl-Cys-Ser-Glu-Glu-Glu-Glu (Ad-4) or tripalmityl-Cys-Ser-Lys-Lys-Lys-Lys (Ad-2).

Example 3

This Example describes the formulation of adjuvants with an antigen.

The Adjuvant BAY R1005, OTH or TPC derivatives were sterile-distilled Water or saline buffer using, for example, a sonicator 2 to 30 minutes long mixed. The adjuvants were on a laboratory balance as stock solutions of 10 mg / ml for BAY R1005, 1 mg / ml for OTH and TPC derivatives were prepared. The stock solution of the synthetic adjuvant was added to an antigen, by 1 mg / ml BAY R1005 or 200 μg / ml To give OTH or TPC derivatives. The antigen can be, for example by methods of peptide synthesis and recombinant techniques, which are not explicitly described in this specification, however in detail set out in the scientific literature and within the level of knowledge of a person of ordinary skill in the art, to be obtained.

Example 4

This Example describes the preparation of adjuvant compositions and immunogenic compositions containing an inorganic adjuvant salt and at least one other adjuvant.

One Synthetic adjuvant can be prepared as described in Example 2 and the inorganic adjuvant salt may be aluminum phosphate clinical grade which may be obtained, for example, from Connaught Laboratories Limited, Toronto, Canada, available is.

The immunogenic composition was prepared by first mixing BAY R1005 with sterile distilled water or saline buffer using a sonicator for two to thirty minutes. Ten ml of the desired amount (1 to 200 μg per ml) of antigen absorbed to 3.1 mg per ml of AlPO ₄ suspension for 1 to 24 hours was added to 1 ml of BAY R1005 stock solution and the solution was gently agitated for 30 Mixed minutes at room temperature.

Preparation of an immunogenic Composition of a synthetic HIV peptide

A BAY R1005 stock solution of 10 mg / ml was prepared as described above. Ten ml of the desired amount (20 to 200 μg per ml) of synthetic HIV peptides (Table 1), absorbed to 3.1 mg per ml of AlPO ₄ suspension for 1 to 24 hours, were then added to 1 ml of BAY R1005 stock solution, and the solution was gently mixed for 30 minutes at room temperature. The synthetic HIV peptide vaccine formulation was stored at 4 ° C for long-term storage. The vaccine formulation was gently mixed for 10 minutes at room temperature before being injected into hosts.

Preparation of a Flu Vaccine Formulation

A BAY R1005 stock solution of 10 mg / ml was prepared as described above. Ten ml of solution containing twenty human doses of Flu split vaccine obtained commercially from Pasteur Merieux SV, Lyon, France, was added to 1 ml of BAY R1005 stock solution. The mixture was vortexed for 10 minutes at room temperature. The emulsified flu vaccine was then mixed with 3.1 mg per ml of AlPO ₄ suspension for 1 to 24 hours and then the vaccine formulation was stored at 4 ° C.

Preparation of RSV Peptide Vaccine Formulation

A BAY R1005 stock solution of 10 mg / ml was prepared as described above. 10 ml of the desired amount (100 μg per ml) of synthetic RSV peptides (Table 1), absorbed to 3.1 mg per ml of AlPO ₄ suspension for 1 to 24 hours, were added to 1 ml BAY R1005 stock solution, and the solution was gently mixed at room temperature for 30 minutes. The synthetic RSV peptide vaccine formulation was stored at 4 ° C.

Preparation of PIV3 vaccine formulation

A BAY R1005 stock solution of 10 mg / ml was prepared as described above. 10 mL of the desired amount (0.3 to 3 micrograms per ml) PIV3 subunit vaccine containing HN and F proteins absorbed onto 3.1 mg per mL of AlPO ₄ suspension for 1 to 24 hours. The PIV3 subunit vaccine was prepared according to published international patent application WO 91/00104 assigned to the assignee hereof. The alum-absorbed PIV3 antigens were added to 1 ml BAY R1005 stock solution and the solution was gently mixed for 30 minutes at room temperature. The PIV3 vaccine formulation was stored at 4 ° C.

Preparation of Flu BHA vaccine formulation

A BAY R1005 stock solution of 10 mg / ml was prepared as described above. 10 ml of the desired amount (0.5 to 10 μg per ml) Flu BHA subunit vaccine was absorbed to 3.1 mg per ml AlPO ₄ suspension for 1 to 24 hours. The Flu BHA subunit vaccine, prepared according to Brand and Skehel (Nature New Biol. 1972, 238: 145-147), was prepared by Dr. med. D. Burt of Connaught Laboratories Limited, Willowdale, Ont. Canada, provided. The alum-absorbed Flu-BHA was added to 1 ml BAY R1005 stock solution and the solution was gently mixed for 30 minutes at room temperature. The Flu BHA vaccine formulation was stored at 4 ° C.

Preparation of pertussis toxoid vaccine formulation

A BAY R1005 stock solution of 10 mg / ml was prepared as described above. 10 ml of the desired amount (1 to 20 μg per ml) of pertussis toxoid, prepared according to Tan et al., US Pat. No. 4,997,915 assigned to the owner thereof, was added to 3.1 mg per ml of AlPO ₄ . Suspension absorbed for 1 to 24 hours. The alum-absorbed pertussis toxoid was added to 1 ml BAY R1005 stock solution and the solution was gently mixed for 30 minutes at room temperature. The pertussis toxoid vaccine formulation was stored at 4 ° C.

Example 5

This Example describes the general immunization protocol which for testing adjuvant compositions and immunogenic compositions was used.

Guinea pig immunizations

Three guinea pigs were used. The animals were pre-bled on day 0 and then given 500 μl of the formulation containing the desired amount of antigen, 1.5 mg of ALPO ₄ and 500 μg of the immunomodulators BAY R1005 or its derivatives on day 1, 14 and 29 im injected. Blood samples were obtained on day 28 and the animals were bled on day 42. Antibody titers were determined for peptide-specific IgG antibodies using an antigen-specific enzyme-linked immunosorbent assay (ELISA). Functional antibody responses were measured using virus neutralization and / or haemagglutination inhibition (HAI) assays, and for pertussis toxoid, the ability of antisera to inhibit induced CHO cell clustering was determined.

Mouse immunizations

Three to five mice were used. The animals were pre-bled on day 0, and then 200 μl of the formulation containing the desired amount of antigen, 0.6 mg AlPO ₄ and 200 μg adjuvant BAY R1005 or its derivatives on day 1, 14 and 29 ip injected. Blood samples were obtained on day 28 and the animals were bled on day 42. Antibody titers were determined for peptide-specific IgG antibodies using an antigen-specific enzyme-linked immunosorbent assay (ELISA). Functional antibody responses were determined using virus neutralization and / or haemagglutination inhibition (HAI) assays.

Example 6

This Example describes an analysis of the immune response to immunogenic Compositions.

Antigen-specific ELISAs

Microtiter wells (Nunc-Immunoplate, Nunc, Denmark) were probed with 200 ng of purified antigen (PT, HA, PIV3, HN and F) or 500 ng of individual peptides in 50 μl of coating buffer (15 mM Na ₂ CO ₃ , 35 mM NaHCO ₃ , pH 9.6) for 16 hours at room temperature. The plates were then blocked with 0.1% (w / v) BSA in phosphate buffered saline (PBS) for 30 minutes at room temperature. Serial sera antisera were added to the wells and incubated for one hour at room temperature. After removal of the antisera, the plates were washed five times with PBS containing 0.1% (w / v) Tween-20 and 0.1% (w / v) BSA. F (ab ') ₂ fragments of goat anti-rabbit, guinea pig, mouse or human IgG antibodies conjugated to horseradish peroxidase (Jackson ImmunoResearch Labs Inc., PA) were diluted (1/8000 ) with washing buffer and the microtiter plates added. After one hour of incubation at room temperature, the plates were washed five times with the washing buffer. The plates were then developed using the substrate tetramethylbenzidine (TMB) in H ₂ O ₂ (ADI, Toronto). The reaction was quenched with 1N H ₂ SO ₄ and the optical density measured at 450 nm using a Titretek Multiskan II (Flow Labs., Virginia). Two irrelevant pertussis toxin peptides NAD-S1 (19 residues) and S3 (123-154) (32 residues) were included as negative controls in the peptide-specific ELISAs. Assays were performed in triplicates and the reactive titer of each antiserum was defined as the dilution which consistently showed a two-fold increase in absorbance value over that obtained by the negative controls.

Haemagglutinationsinhibierungs (HAI) assays

The Assay is based on the assets of antisera, either influenza virus or PIV3 can, guinea pig red blood cells (PIV3) or chicken (flu virus) to agglutinate. Red blood cells become the wells of a 96-well microtiter plate, which serial dilutions added to antisera and a constant amount of virus. After incubation, the HAI titer is read by the degree Haemagglutinininhibition is determined. The lowest dilution of the Antisera that blocks haemagglutination is the endpoint titer. A reference sample with known HAI is run in parallel.

Virus neutralization assays

The Assay is based on the assets of antisera to inhibit PIV3 growth in Vero cells. To Vero cells grown in wells of a 96-well microtiter plate are serial dilutions of antisera and one constant amount of virus added. After the incubation will The UN titre of each antiserum was read by 50% inhibition of the cytopathic effect caused by PIV3. The lowest dilution antisera blocking the virus-cytopathic effect, is the endpoint titer. A reference sample with a known UN titre is run in parallel.

HIV-1 virus neutralization assay

The ability of antisera to neutralize HIV-1 was determined in a syncytia (polynuclear giant cell) inhibition assay. Ten μl of antiserum in serial dilution was added to the wells of a 96-well tissue culture plate. 5 - 10 x 10 ³ HIV-1 infected CEM cells (in 50 μl) were added to each well. 7 x 10 ⁴ uninfected MOLT-4 cells (in 50 μl) were then added to each well. The plates were then incubated in a CO ₂ incubator overnight. In the samples in which no syncytia had been formed (ie, functional virus-neutralizing antibodies were present in the antiserum), these plates were incubated for an additional 24 hours and then re-assayed for syncytia formation. The number of syncytia was evaluated under an inverted microscope. The lowest dilution of antisera blocking 90% of virus syncytia formation is the endpoint titer. A reference sample with a known virus neutralizing titer was run in parallel.

Example 7

This Example describes the protocol used to generate antigen-specific Through T cell lines immunogenic preparations of the present invention can be used.

BALB / c (H-2 ^d ) mice purchased from Charles River Animal Farm (Montreal, Canada) were individually subcutaneously emulsified with the desired amount of antigen (1-100 μg) either with BAY R1005 or its derivatives, primed. The animals were boosted twice with the same dose of immunogen at 3 week intervals. Ten days after the last boost, the spleens of the immunized mice were removed. Spleen cells were seeded at 6 x 10 ⁵ cells per well in a final volume of 200 μl RPMI 1640 medium (Flow Lab.) Supplemented with 10% heat-inactivated fetal calf serum (Gibco), 2 mM L-Glutamine (Flow Lab.), 100 U / ml) penicillin (Flow Lab.) and 5 x 10 ^-5 M 2-mercaptoethanol (Sigma), in the presence of various concentrations (1, 10 and 100 μg per ml) of antigen in 96-well plates (Nunc, Denmark) , Cultures were maintained in a humidified incubator in the presence of 5% CO ₂ / air. Triple cultures were performed for each concentration of antigen. Five days later, 150 μl of 10% rat concanavalin A culture supernatant diluted in culture medium was added to the microtiter plate wells as a source of interleukin-2 (IL-2) to expand antigen-specific T cells. Six days later, 150 μl of supernatant was removed from each microculture, and 150 μl of fresh culture supernatant containing IL-2 was added to further expand and maintain the viability of antigen-specific T cells. After a further 6-day incubation, the cells were washed three times, each time with 200 μl of culture medium.

Each set of cultures was then stimulated with the appropriate concentrations (1, 10 and 100 μg per ml) of the antigen in the presence of 2 x 10 ⁵ irradiated (1500 rad) BALB / c spleen cells in a final volume of 200 μl culture medium. Sixty μl of supernatant was then removed from each microculture. The supernatants from all triplicate cultures were then pooled. All supernatants were then assayed for IL-2, interleukin-4 and interferon-gamma (IFN-γ) using mouse IL-2 and IL-4 ELISA kits, respectively, purchased from Endogen Inc. (MA, USA ), certainly. An assay of IFN-γ can be performed using a mouse IFN-γ ELISA kit sold by Genzyme Corporation (MA, USA). Test culture supernatants can be tested at a 1 in 5 dilution according to the manufacturer's instructions.

Example 8

This Example describes the covalent attachment of BAY R1005 to a Peptide.

N- (glutarylacylamido-2-deoxy-β-D-glucopyranosyl) -N-alkylcarboxamide, which can be used to covalently attach BAY R1005 derivatives to either a peptide or protein, was synthesized as follows. To a solution of N- (2-amino-2-deoxy-β-D-glucopyranosyl) -N-alkyl-carboxamide (2 mmol) in dioxane (28 ml) was added diisopropylethylamine (8 mmol) and glutaric anhydride (12 mmol ) added. The reaction mixture was stirred overnight at room temperature under argon. Ammonium hydroxide (28 ml) was added to the mixture, and then it was stirred for an additional 18 hours. The product was formed as a precipitate, which was filtered, and then redissolved in a solution containing water and t-butanol (2: 1). The solution was then acidified by the gradual addition of acetic acid (4 ml). The solution was then lyophilized to give the product in 73% yield, mass spectral analyzes (FAB-HRMS) of N- (glutarylacylamido-2-deoxy-β-D-glucopyranosyl) -N-alkyl-carboxamide, Ca ₃ H ₈₃ N ₂ O ₈ , calculated 755.6149; and found 755.6129.

resin-bound side chain protected CLTB-36 peptide was synthesized using F-moc chemistry as follows. two hundred to 500 mg of resin containing the N-Fmoc-protected first amino acid residue wore, were placed in a reaction tube. The resin was washed four times with DMF, then with a 50% solution of piperidine in DMF (5 ml during 1 minute) and washed with a 50% solution of piperidine in DMF (10 ml while 9 minutes) deprotected. The resin was then washed with DMF (5 times, 10 ml each). The Peptide resin was then added with 5 equivalents the desired Fmoc protected Amino acids, activated with phosphonium hexafluorophosphate ester (pfpe) in the presence coupled from DIEA at room temperature for 2 to 3 hours and with DMF washed (5 times, 10 ml each). After the last DMF washing step became an aliquot for taken a ninhydrin test. If the test was negative, you go to Step 1 to pair the next Amino acid. If the test was positive or slightly positive, the coupling and DMF washes repeated.

The N- (glutarylacylamido-2-deoxy-β-D-glucopyranosyl) -N-alkyl-carboxamide was conjugated to the synthetic peptide CLTB-36 as follows: N-hydroxysuccinimide (335 μg, 1.12 eq.) Became a solution, containing the glycolipid, prepared as described above (2,0 mg), and dicyclohexylcarbodiamide dissolved in dichloromethane (2 ml) contained, added. The resulting mixture was then heated for 4 Stirred hours at room temperature. The dicyclohexylurea was filtered and the resulting Filtrate was evaporated to dryness. The dried active Ester was redissolved in DMF (0.1 ml) to become a peptide-resin which contained side-chain protected CLTB-36, and although in a molar ratio from 1.1 to 1. The reaction mixture was left overnight at room temperature touched. The excess reagents were added filtered off and the peptide-resin was washed with 4 x 10 ml dichloromethane. After drying, the peptide-resin was treated with TFA to give lipidated Remove CLTB-36 from the resin. The crude lipidated peptide was further purified by HPLC a C4 Vydac column (1 × 35 cm) with an acetonitrile gradient of 20 to 60%, applied while 40 minutes at a flow rate of 2 ml / minute, cleaned. The amino acid composition analyzes of lipidated CLTB-36 that were performed on a Waters Pico-Tag system, were in good agreement with their theoretical compositions, and the presence of fatty acids was determined by GC analyzes of the acid hydrolyzate approved.

The N- (glutarylacylamido-2-deoxy-β-D-glucopyranosyl) -N-alkyl-carboxamide was conjugated to recombinant Flu NP protein as follows:
N-hydroxysuccinimide (335 μg, 1.12 eq.) Was added to a solution containing the glycolipid (2.0 mg) prepared as described above and dicyclohexylcarbodiamide dissolved in dichloromethane (2 ml). The resulting mixture was then stirred for 4 hours at room temperature. The dicyclohexylurea was filtered and the resulting filtrate was evaporated to dryness. The dried active ester (2.25 mg) was redissolved in DMF (0.1 ml) and added to a protein solution (2 mg of rNP1 protein dissolved in 2 ml of 25 mM phosphate buffer, pH 7.8) in a molar ratio of 20 : 1 added. The reaction mixture was stirred overnight at 4 ° C and then dialyzed against 4 x 41 of the phosphate buffer. Incorporation of N- (glutarylacylamido-2-deoxy-BD-glucopyranosyl) -N-alkyl-carboxamide into recombinant Flu-NP was determined and confirmed using gas chromatography (GC) for lipid analysis of the acid hydrolyzate (6N HCl for 2 hours at 110 ° C) of the modified protein.

The The present invention has been exemplified by reference to particular Examples and embodiments explained.

SUMMARY OF THE DESCRIPTION

TABELLE 2 Meerschweinchen-Antikörper-Antworten auf synthetische Peptide, welche mit unterschiedlichen Adjuvantien formuliert sind

• 1 Durchschnittlicher Titer von drei Meerschweinchen, welche drei Injektionen erhielten.
• 2 Alle Peptidimmunogene außer CLTB-36 werden mit 100 μg Peptid pro Dosis, formuliert mit unterschiedlichen Adjuvantien, verwendet. CLTB-36 wird mit 20 μg Peptid pro Dosis verwendet.
• 3 Nicht durchgeführt.

TABELLE 3 Meerschweinchen-Antikörper-Antworten auf synthetische Peptide, welche mit unterschiedlichen Adjuvantien formuliert wurden

• 1 Durchschnittstiter von 3 Meerschweinchen, die drei Injektionen erhielten.
• 2 Alle Peptid-Immunogene werden mit 100 μg Peptid pro Dosis verwendet, formuliert mit unterschiedlichen Adjuvantien.

TABELLE 4 ANTIKÖRPERANTWORT AUF IN UNTERSCHIEDLICHEN ADJUVANTIEN FORMULIERTES CLTB-36

• 1 Gruppen von Meerschweinchen (n = 3) wurden mit 20 μg CLTB-36, emulgiert in BAY R1005 und/oder absorbiert auf Alumen, am Tag 1 immunisiert. Boosterimmunisierungen wurden nach 2, 4 und 6 Wochen mit den gleichen Antigenen vorgenommen. Blutproben wurden alle 2 Wochen nach der dritten Injektion gesammelt. Seren wurden mittels peptidspezifischen ELISAs, Virus-Neutralisations- und Syyncytia-Bildungs-Inhibitions-Assays analysiert.
• 2 Das Peptid CLTB-56 ist das B-Zell-Epitop von CLTB-36 und besitzt die Aminosäuresequenz NKRKRIHIGPGRAFYTTKN (SEQ ID NR.: 16).
• 3 100 μg CLTB-36, das auf Alumen absorbiert war, wurden eingesetzt.

To summarize this description, the present invention provides novel adjuvant compositions capable of eliciting an enhanced immune response to antigens and novel compounds comprising antigen covalently linked to a glycolipid analog. Table 1 SYNTHETIC PEPTIDES USED IN THE STUDIES

Guinea pig antibody responses to synthetic peptides formulated with different adjuvants

• 1 Average titre of three guinea pigs receiving three injections.
• 2 All peptide immunogens except CLTB-36 are used with 100 μg of peptide per dose, formulated with different adjuvants. CLTB-36 is used with 20 μg of peptide per dose.
• 3 Not performed.

Guinea pig antibody responses to synthetic peptides formulated with different adjuvants

• 1 average titre of 3 guinea pigs receiving three injections.
• 2 All peptide immunogens are used with 100 μg of peptide per dose, formulated with different adjuvants.

TABLE 4 ANTIBODY RESPONSE TO CLTB-36 FORMULATED IN DIFFERENT ADJUVANTIES

• 1 group of guinea pigs (n = 3) were immunized with 20 μg of CLTB-36 emulsified in BAY R1005 and / or absorbed on alum, on day 1. Booster immunizations were done after 2, 4 and 6 weeks with the same antigens. Blood samples were collected every 2 weeks after the third injection. Sera were analyzed by peptide-specific ELISAs, virus neutralization and syyncytia formation inhibition assays.
• 2 The CLTB-56 peptide is the B-cell epitope of CLTB-36 and has the amino acid sequence NKRKRIHIGPGRAFYTTKN (SEQ ID NO: 16).
• 3 100 μg of CLTB-36 absorbed on alum were used.

Claims (18)

Adjuvant composition for modulating a Immune response to an antigen administered to a host, the composition being characterized by: (a) a inorganic adjuvant salt; and (b) at least one other Adjuvant, which consists of the group consisting of a glycosylamide, a Octadecyl ester or an amino acid and a lipopeptide is. A composition according to claim 1, wherein the inorganic Adjuvant salt from the group consisting of aluminum hydroxide, aluminum phosphate, Calcium phosphate, zinc hydroxide and calcium hydroxide is selected. A composition according to claim 1 or 2, wherein the amino acid an aromatic amino acid is. A composition according to claim 3, wherein the aromatic amino acid Tyrosine is. A composition according to claim 1 or 2, wherein said Lipopeptide a synthetic analogue of the N-terminal part of a Lipoprotein from a Gram-negative bacterium. A composition according to claim 1 or 2, wherein said Lipopeptide Tripalmityl-S-glyceryl-cysteinylseryl-serine or N-palmityl-S- (2,3-palmityloxy) -2- (RS) -propyl- [R] -cysteine is. A composition according to claim 1 or 2, wherein the glycosylamide has the formula:

in which R ^{1 represents} hydrogen or a saturated or mono- or polyunsaturated alkyl radical having up to 50 carbon atoms; X represents -CH ₂ -, -O- or -NH-, R ^{2 represents} hydrogen or a saturated or mono- or polyunsaturated alkyl radical having up to 50 carbon atoms; R ³ , R ⁴ and R ^{5 are} independently hydrogen or SO ₄ ^2- or PO ₄ ^2- or another negatively charged radical, or -CO-R ⁶ wherein R ^{6 is} an alkyl radical of up to 10 carbon atoms; R ^{7 is} L-alanyl, L-alpha-aminobutyryl, L-arginyl, L-asparaginyl, L-aspartyl, L-cysteinyl, L-glutamyl, L-glycyl, L-histidyl, L-hydroxypropyl, L-isoleucyl, L- Leucyl, L-lysyl, L-methionyl, L-ornithinyl, L-phenylalanyl, L-prolyl, L-seryl, L-threonyl, L-tyrosyl, L-tryptophanyl and L-valyl or their D-isomers; and pharmaceutically acceptable salts thereof. A composition according to claim 7, wherein the glycosylamide N- (2-deoxy-2-L-leucylamino-β-D-glucopyranosyl) -N-octadecyldodecanamidacetat is. A composition according to claim 8, wherein the inorganic Salt is aluminum phosphate. A composition according to any one of claims 1 to 9, further comprising at least one antigen for triggering a Immune response in a host. A composition according to claim 10, wherein the antigen from the group consisting of microbial pathogens, bacteria, Viruses, proteins, glycoproteins, lipoproteins, peptides, glycopeptides, Lipopeptides, toxoids, carbohydrates and tumor-specific antigens selected is. A composition according to claim 11, wherein the antigen a protein, glycoprotein or lipoprotein, or a peptide, glycopeptide or lipopeptide containing an amino acid sequence which an antigenic determinant of HIV, rubella virus, respiratory Syncytial virus, Bordetella pertussis, Haemophilus influenzae or Streptococcus pneumoniae corresponds. The composition of claim 12, wherein the peptide contains an amino acid sequence as shown in Table 1. A composition according to claim 10, wherein the antigen comprises a toxoid which is the pertussis toxoid. The composition of claim 11, wherein the protein Influenza hemagglutinin or a subunit of a human parainfluenza virus. Adjuvant composition characterized by inorganic adjuvant salt and at least one other adjuvant, that from the group consisting of a glycosylamide, an octadecyl ester or an amino acid and a lipopeptide is for use in modulating an immune response to a Antigen administered to a host. Use of an adjuvant composition characterized by an inorganic adjuvant salt and at least one other Adjuvant selected from the group consisting of a glycosylamide, an octadecyl ester an amino acid and a lipopeptide is using an antigen to make a drug for modulation an immune response to an antigen administered to a host. Kit for the preparation of an immunogenic composition, full: (a) agents containing an inorganic adjuvant salt; (B) Agent containing at least one other adjuvant consisting of the group from a glycosylamide, an octadecyl ester or an amino acid and a lipopeptide selected is; (c) agents containing at least one antigen; and (D) Means for combining the inorganic adjuvant salt, at least another adjuvant and at least one antigen, for production the immunogenic composition.